Candidatus Methanogranum caenicola: a novel methanogen from the anaerobic digested sludge, and proposal of Methanomassiliicoccaceae fam. nov. and Methanomassiliicoccales ord. nov., for a methanogenic lineage of the class Thermoplasmata

Abstract

The class Thermoplasmata harbors huge uncultured archaeal lineages at the order level, so-called Groups E2 and E3. A novel archaeon Kjm51a affiliated with Group E2 was enriched from anaerobic sludge in the present study. Clone library analysis of the archaeal 16S rRNA and mcrA genes confirmed a unique archaeal population in the enrichment culture. The 16S rRNA gene-based phylogeny revealed that the enriched archaeon Kjm51a formed a distinct cluster within Group E2 in the class Thermoplasmata together with Methanomassiliicoccus luminyensis B10(T) and environmental clone sequences derived from anaerobic digesters, bovine rumen, and landfill leachate. Archaeon Kjm51a showed 87.7% 16S rRNA gene sequence identity to the closest cultured species, M. luminyensis B10(T), indicating that archaeon Kjm51a might be phylogenetically novel at least at the genus level. In fluorescence in situ hybridization analysis, archaeon Kjm51a was observed as coccoid cells completely corresponding to the archaeal cells detected, although bacterial rod cells still coexisted. The growth of archaeon Kjm51a was dependent on the presence of methanol and yeast extract, and hydrogen and methane were produced in the enrichment culture. The addition of 2-bromo ethanesulfonate to the enrichment culture completely inhibited methane production and increased hydrogen concentration, which suggested that archaeon Kjm51a is a methanol-reducing hydrogenotrophic methanogen. Taken together, we propose the provisional taxonomic assignment, named Candidatus Methanogranum caenicola, for the enriched archaeon Kjm51a belonging to Group E2. We also propose to place the methanogenic lineage of the class Thermoplasmata in a novel order, Methanomassiliicoccales ord. nov.

Fig. 1

Phylogenetic affiliation of the enriched archaeon Kjm51a within Group E2 in the class Thermoplasmata on the basis of the 16S rRNA gene sequences. The tree was constructed using the neighbor-joining method. Solid circles at branching nodes indicate supporting probabilities above 95% by all the phylogenetic analysis methods (NJ, ML, and Bayesian), and open circles indicate probabilities above 85% by two or more analyses. Bar, 0.02 substitutions per nucleotide position.

Fig. 2

Deduced McrA amino acid sequence-based phylogeny showing the phylogenetic relationships among the enriched archaeon Kjm51a, its related archaeon and environmental clones, and other known methanogens. Solid circles at branching nodes indicate supporting probabilities above 95%, and open circles indicate probabilities above 80%. Bar, 0.02 substitutions per nucleotide position.

Fig. 3

Epifluorescence micrographs of in situ hybridization of the enrichment archaeon Kjm51a grown on MYB medium for a week. The same microscopic field is shown after hybridization with a Kjm51a-specific probe (red), an archaeal probe ARC915 (green), and a bacterial probe EUB338 (blue). A, blue color; B, green color; C, merge of red, green and blue colors; D, red color. Bars, 10 μm.

Fig. 4

Methane and hydrogen production in the enrichment culture obtained from anaerobic sludge. Filled bars, methane; open squares, hydrogen. Data points and bars are the means and standard deviations, respectively (n=3). Abbreviations: MYB, MYB medium supplied with methanol in YB medium; YB, YB medium; MYB+BES, MYB medium supplied with BES; ND, not detected.